It has been suggested that two categories of Treg can be identified by phenotypical and functional characteristics. "Innate" and "adaptive" Treg would cooperate in limiting potentially noxious inflammatory processes. This regulatory function may be impaired in autoimmunity. Its restoration could provide novel therapeutic approaches. This project aims to unravel the role, which may pertain to Treg function in induction of tolerance to an antigenic peptide (dnaJP1) in rheumatoid arthritis (RA). 105 samples from RA patients treated in the context of a Phase 1 (completed) and a Phase II (ongoing) clinical trial with dnaJP1 or placebo have already been collected at the beginning of treatment and at monthly intervals. We will test whether mucosal tolerization to a peptide is associated with emergence of cells with a regulatory phenotype. We will also determine if a proportion of Treg are dnaJP1-specific, and if numbers and functional characteristics of these cells change as a consequence of immunotherapy. The specific aims are: Specific Aim 1: To characterize "innate" and "adaptive" Treg in serial samples obtained from a tolerization trial in rheumatoid arthritis and to explore their functional role in the tolerization process. SA1a: Peripheral blood mononuclear cells (PBMC), synovial fluid mononuclear cells (SFMC) and T cells obtained from synovial membranes of RA patients will be evaluated by FACS analysis for phenotypical markers characteristic of T regulatory T cells. In particular, levels of CD25, CD4, CTLA4 and CCR4 will be studied. "Innate" and "adaptive" T cells will be differentiated based on a set of phenotypical and functional variables, including levels of CD25 expression and quantification on sorted cells by real time PCR (TaqMan) gene expression of several molecules putatively involved in Treg function. These genes will include IL-10, TGF beta, IL-4, FOXP3, CTLA. Samples obtained will be also tested in in vitro studies to explore regulatory properties of "innate" and "adaptive" Treg on T cell responses to recall antigens as well as to antigens (gp39, dnaJP1 and p205) putatively involved in the pathogenic process. SA1b: Clinical information will be compared with immunological data Specific Aim 2: To investigate whether some Treg have specificity for dnaJpl and whether functional and phenotypical characteristics of dnaJP1-specific Treg are associated with the course of the tolerization process. Functional characteristics of sorted cells will be evaluated by TaqMan. [unreadable] [unreadable]